Immunoglobulin G (IgG) antibodies consist of two heavy chains (Hc) and two light chains (Le). Monoclonal antibodies (mAbs) have been increasingly developed as medicines over the last three decades and represent the fastest growing class of therapeutic agents (Walsh, Biopharmaceutical Benchmarks 2014; Reichert, mAbs 2:84-100, 2010). This has been enabled by the development of technologies for antibody discovery, such as phage and ribosome display and transgenic mice with human antibody repertoires (Strohl, Curr. Drug Discov. Technol. 11:1-2, 2014). In a biopharmaceutical environment, antibodies can be generated by molecular manipulation of DNA and transfection of mammalian host cells to produce specifically targeted biotherapeutics. The lead antibody molecules from the discovery process need to be produced in significant quantities for use in development and for clinical applications. Typically, the large scale production of recombinant antibodies employs stable recombinant mammalian cell lines such as CHO or NS0, owing to the ability of these host cells to correctly fold and assemble antibodies, and to perform the required post-translational modifications (Walsh, 2014). Generally, the host cell lines are engineered by transfecting and selecting for the integration of expression plasmids encoding the antibody genes of interest. For example, Chinese Hamster Ovary (CHO) cells can be transfected with DNA containing coding regions for Lc and Hc variable and constant domains. This DNA stably integrates into the CHO host cell genome, and is then transcribed and translated to produce the Lc and Hc polypeptides, each of which contains a secretory leader sequence.
The secretory leader sequences act as a “postcode” for the peptides, directing the nascent polypeptides from the cytosol through translocons to the endoplasmic reticulum (ER). More particularly, as the nascent polypeptide emerges from the ribosome in the cytosol, the signal peptide binds to the signal recognition particle (SRP) and this complex is targeted to the translocon in the endoplasmic reticulum (Nyathi et al., Molec. Cell Res. 1833:2392-2402, 2013). Then, as the polypeptide is translocated from the cytosol across the membrane into the ER, the signal peptide itself is cleaved off by the signal peptidase so that the signal peptide is not part of the mature protein. The Hc and Lc polypeptides are processed into the final antibody protein product by the cellular machinery, and the antibody is secreted into the cell supernatant.
Eukaryotic signal peptides are characterized by structural homology, rather than significant sequence conservation, having a three-domain structure: a positively charged N-terminal domain (N-domain), a central hydrophobic region (h-domain) and a more polar C-terminal region (C-domain) (Von Heijne, J. Molec. Biol. 184:99-105, 1985). The h-domain is a major feature for SRP recognition and binding, and the hydrophobicity is important for translocation across the ER membrane (Nilsson et al., J. Molec. Biol. 427:1191-1201, 2015). The C-domain defines the cleavage site and must fulfil a “−3, −1 rule” (Von Heijne, J. Molec. Biol. 173:243-251, 1984; Von Heijne, Eur. J. Biochem. 133:17-21, 1983), where the −1 position of the signal peptide (i.e., the last residue before the cleavage site) must be occupied by A/S/G/C/T/Q amino acid residues, and must not have aromatic (F/H/W), charged (D/Q/K/R), or large polar residues (N/Q) in position −3, as well as no P from −3 to +1. A similar signal peptidase recognition site, A-X-B, just before the cleavage site, has also been postulated (Perlman et al., J. Molec. Biol. 167:391-409, 1983). Position A consists of A/G/S residues or the larger aliphatic amino acids L/V/I, and position B is occupied by A/G/S. The amino acid occupying −1 of the signal peptide is critical for the site of signal peptidase cleavage (Folz et al., J. Biol. Chem. 263:2070-2078, 1988). In addition, the identity of amino acid residues upstream of the −1 and −3 positions and the h-/C-domain junction influences the site and efficiency of cleavage (Nothwehr et al., J. Biol. Chem. 264:3979-3987, 1989). The position of the junction between the N- and h-domains also influences the cleavage site (Nothwehr et al., J. Biol. Chem. 265:21797-21803, 1990).
It is essential that therapeutic antibodies are produced consistently; transfected cell lines must produce protein that is stable and has reproducible product quality attributes. For instance, it is undesirable to have impurities (i.e., any substance that is not part of the final, intact monoclonal antibody or final formulation buffer) in the final therapeutic protein product. Impurities can lead to instability of the product, and they have the potential to cause immunogenicity, decrease the potency of the product and/or have off-target effects, which could compromise patient safety. Impurities can be removed by downstream processing of the raw cell culture harvest; however, this process is expensive in both time and cost of goods. Therefore, it is beneficial to ensure there are as few impurities present in the final culture harvest as possible before downstream processing. It is also difficult to both detect and remove impurities that closely resemble the IgG product. As such, it is desirable for these product-related variants to be minimized in the culture harvest.
The signal peptide cleavage process is generally highly efficient for antibodies, producing a high proportion of correctly cleaved heavy and light chain polypeptides. However, there are some documented cases where the cleavage site is variable resulting in truncation or extension of mAb heavy and light chains (Ambrogelly et al., mAbs 4:701-709, 2012; Kotia et al., Anal. Biochem. 399:190-195, 2010; Ying et al., Immunol. Lett. 111:66-68, 2007; Shaw et al., Molec. Immunol. 29:525-529, 1992). We have used the murine heavy chain signal peptide sequence (Persic et al., Gene 187:9-18, 1997; Orlandi et al., Proc. Natl. Acad. Sci. USA 86:3833-3837, 1989) for secretion of heavy and light chains for a wide variety of human antibodies and have observed correct processing of the signal peptide resulting in a highly homogeneous N-terminal sequence. Here we describe the characterization and prevention of a recombinant human IgG light chain truncation, not previously detailed in the literature, that is associated with the combination of a murine heavy chain signal peptide sequence with lambda light chains carrying an N-terminal SYE amino acid motif.
Specifically, during development of a therapeutic IgG (MEDI8490), disclosed in U.S. patent application Ser. No. 14/435,520 herein incorporated by reference in its entirety, we observed that about 3-8% of the final antibody product contained a truncated Lc peptide. The truncated Lc was missing three amino acids at its N-terminus: serine-tyrosine-glutamic acid (SYE). The truncated Lc was detected by liquid chromatography-mass spectrometry (LC-MS) analysis of deglycosylated IgG, and was confirmed by reduced peptide mapping analysis. The truncated Lc is considered to be a product-related variant. In order to increase the homogeneity of the product and to reduce product development risks, we investigated ways to prevent the Lc SYE truncate from being produced. Two different protein engineering solutions were explored: (1) alteration of the Lc N-terminal SYE amino acid sequence to other alternatives; (2) changes to the secretory leader peptide sequence. We have shown that both of these solutions are effective for preventing N-terminal Lc truncation in the MEDI8490 antibody and other IgG proteins. This demonstrates that these are broadly applicable solutions to this issue for IgG production.